Karyotype and Chromosome Analysis

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Questions and Answers

What does the speed of migration of charged substances in electrophoresis depend on?

Temperature and pressure of the solution

Concentration of the electric field

Purity of the supporting medium

Net charge, size, and shape of the substance (correct)

Which statement is true about the migration of charged molecules in an electric field?

Molecules migrate towards the electrodes of opposite charge (correct)

Molecules with a net negative charge move towards the cathode

All molecules migrate towards the anode regardless of charge

Molecules with a net positive charge move towards the anode

Who is recognized as the father of electrophoresis?

Richard Feynman

Arne Tiselius (correct)

Albert Einstein

Michael Faraday

In which year did Arne Tiselius develop the zone method for purifying biomolecules?

1930 Signup and view all the answers

What is the primary purpose of electrophoresis?

To separate electrically charged substances in a mixture Signup and view all the answers

What defines a karyotype?

The complete set of chromosomes in a species. Signup and view all the answers

Which specimen is commonly used for karyotyping?

Peripheral blood lymphocytes Signup and view all the answers

What is C-banding primarily useful for?

Determining the presence of dicentric and pseudodicentric chromosomes. Signup and view all the answers

What happens during T-banding?

Staining diminishes except at telomeres. Signup and view all the answers

Which technique would be used to visualize only the centromeric regions of chromosomes?

C-banding Signup and view all the answers

Which method involves the staining of chromosomes to view specific light and dark bands?

Chromosomal banding Signup and view all the answers

What type of specimens are preferred for obtaining dividing cells for karyotyping?

Spontaneously proliferating cells Signup and view all the answers

What is the main purpose of polymerase chain reaction in genetics?

To amplify specific DNA sequences. Signup and view all the answers

What is the initial temperature for the denaturation step in a standard PCR cycling?

94 °C to 98 °C Signup and view all the answers

During which step do oligonucleotide primers attach to complementary sequences?

Annealing Signup and view all the answers

What is the purpose of using reverse transcriptase in modified nucleic acid amplification techniques?

To convert RNA into cDNA Signup and view all the answers

What temperature range is typically used for the annealing step in PCR?

50 °C to 60 °C Signup and view all the answers

What is monitored in real-time PCR (qPCR)?

Amplification of a targeted DNA molecule Signup and view all the answers

What occurs during the extension step of PCR?

DNA polymerase synthesizes new DNA strands Signup and view all the answers

What is the final temperature for the final extension in a standard PCR process?

70 °C to 80 °C Signup and view all the answers

What is the primary advantage of multiplex PCR?

It amplifies multiple DNA sequences simultaneously Signup and view all the answers

What role does the buffer system play in electrophoresis?

Conducts electricity for running the buffer Signup and view all the answers

Which gel type is primarily used for separating large molecules?

Agarose Gel Signup and view all the answers

How is the separation of biomolecules achieved in gel electrophoresis?

Through molecular sieving based on size Signup and view all the answers

In which method can gel electrophoresis be performed vertically?

Vertical Gel Electrophoresis Signup and view all the answers

What is a significant difference between agarose and polyacrylamide gels?

Agarose is less toxic than polyacrylamide Signup and view all the answers

What happens during the process of running the gel until band separation occurs?

Unique banding patterns form based on molecular size Signup and view all the answers

How can staining be performed on agarose gels?

Both before and after pouring the gel Signup and view all the answers

Why is it essential to view DNA on a UV light box after gel electrophoresis?

To visualize the migration of DNA fragments Signup and view all the answers

What is the primary purpose of whole genome sequencing?

To determine the order of bases in an organism's genome. Signup and view all the answers

What technique was developed in 1979 to assist in whole genome sequencing?

Shotgun Sequencing Technique. Signup and view all the answers

What role does DNA bar-coding serve in whole genome sequencing?

To identify which sheared DNA belongs to which bacteria. Signup and view all the answers

Which step in whole genome sequencing directly follows DNA shearing?

DNA bar-coding. Signup and view all the answers

What is one advantage of whole genome sequencing in healthcare?

It helps create personalized medical treatment plans. Signup and view all the answers

Why is it important to identify differences in bacterial sequences during data analysis?

To gauge how closely related the bacteria are. Signup and view all the answers

What size of DNA fragments are typically managed in Sanger sequencing?

100-1000 base pairs. Signup and view all the answers

What characterizes the whole genome sequencing process?

Analyzing the entire genomic sequence in one process. Signup and view all the answers

Study Notes

Karyotype

A karyotype is the complete set of chromosomes in a specie.
It describes the number of chromosomes and their appearance under a light microscope.
It is a photographic representation of stained metaphase spread where chromosomes are arranged by decreasing length.
Specimens with spontaneously proliferating cells include bone marrow, lymph nodes, solid tumors, and chorionic villi.
Peripheral blood lymphocytes, tissue biopsies, and amniotic fluid samples are cultured to obtain dividing cells.
Lymphocytes usually require the addition of a mitotic stimulant.
Specimen selection for chromosome analysis depends on clinical indications and whether the diagnosis is prenatal or postnatal.

Chromosome Staining & Banding

A chromosome band is a distinguishable part of a chromosome.
It is comprised of alternating light and dark stripes.
Banding and staining techniques can be categorized into two groups: those that produce specific alternating bands along the length of each chromosome and those that stain only a specific region.

C-Banding (Constitutive Heterochromatin Banding)

Useful for determining the presence of dicentric and pseudodicentric chromosomes.
Also useful for studying marker chromosomes and polymorphic variants.
It selectively stains the constitutive heterochromatin around the centromeres.
It stains inherited polymorphisms present on chromosomes 1, 9, and 16, and the distal long arm of the Y chromosome.

T-Banding (Telomere Banding)

A harsher treatment of chromosomes diminishes staining except at the heat-resistant telomeres.

Polymerase Chain Reaction (PCR)

Denaturation of dsDNA template occurs at 94℃.
Two oligonucleotide primers anneal at 50-60℃.
Polymerase extension of DNA molecules occurs at 72-74℃.

Standard 3-Step PCR Cycling

Initial denaturation occurs at 94°C to 98°C for 1 minute.
Denaturation occurs at 94°C for 10 to 60 seconds.
Annealing occurs at 50°C to 60°C for seconds.
Final extension occurs at 70°C to 80°C for 5 minutes.
Cycles are typically repeated 25-35 times.
Most thermal cyclers can pause at 4°C indefinitely at the end of the cycles.

Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)

Developed to amplify RNA targets.
An RNA molecule is converted to a cDNA molecule via reverse transcriptase enzyme.
The cDNA molecule is then utilized as a template sequence for the following PCR reaction.
Its application is to detect RNA expression.

Nested PCR

PCR is performed twice.
The second round uses primers that bind to sequences within the product of the first reaction.
Increases specificity and sensitivity by targeting a smaller and more specific region of the DNA.

Multiplex PCR

Amplifies multiple DNA sequences in a single reaction.
Uses different primer pairs for each target sequence.
Allows for the detection of multiple genetic variations or pathogens simultaneously.

Digital PCR

Quantifies the number of DNA molecules in a sample by partitioning it into thousands of individual reactions.
Each reaction contains a single DNA molecule, which is then amplified and detected.
Provides absolute quantification of DNA targets with high sensitivity and precision.

Quantitative PCR (qPCR) or Real-time PCR

Monitors amplification in real-time.
It monitors the amplification of a targeted DNA molecule during the PCR with use fluorescent dyes or fluorophore-containing DNA probes.

Electrophoresis

A method of separating electrically charged substances in a mixture.
A sample of the mixture is placed on a supporting medium to which an electrical field is applied.
Each charged substance migrates toward the cathode or the anode at a speed that depends on its net charge, size, and shape.
Biomolecules migrate towards electrodes of opposite charge due to electrostatic attraction.

Gel Electrophoresis

A type of electrophoresis where the supporting medium is a gel.
Separation occurs through molecular sieving based on the molecular size of the substances.
The gel material acts as a "molecular sieve".
Widely used for separating proteins and nucleic acids.

Two Methods of Gel Electrophoresis

Horizontal Gel Electrophoresis
Vertical Gel Electrophoresis

Types of Gels

Agarose gel: Polysaccharide extracted from seaweed, casted horizontally, non-toxic, separates large molecules, commonly used for DNA separations, staining can be done before pouring the gel.
Polyacrylamide gel: Cross-linked polymer of acrylamide, casted vertically, potent neurotoxic, separates small molecules, used for DNA or protein separation, staining can be done after pouring the gel.

Whole Genome Sequencing

Determines the order of bases in the genome of an organism in one process.
Uses DNA shearing, bar-coding, whole genome sequencing, and data analysis.
DNA shearing cuts DNA into pieces small enough for the sequencing machine to read.
Scientists add small pieces of DNA tags (bar codes) to identify which DNA piece belongs to which bacteria.
The sequencer identifies the A’s, C’s, T’s, and G’s, or bases, that make up each bacterial sequence and uses bar codes to track which bases belong to which bacteria.
Data analysis uses computer tools to compare bacterial sequences and identify differences.
Allows for personalized disease treatment plans, cancer cell genotyping, and identifying previously unknown genes contributing to disease states.

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Description

This quiz covers essential concepts of karyotyping and chromosome banding techniques. It explores the significance of chromosome appearance, specimen selection, and the application of staining methods to analyze chromosomal structures. Perfect for students delving into genetics and cytogenetics.